BRAIN RESEARCH

ELSEVIER Brain Research 714 (1996) 118-124

Research report

Effects of Alzene and tacrine on water maze reference and working memory function in medial septal-lesioned rats

P. Riekkinen Jr. a, *, K.L. Mettinger b, M. Karukin b, M. Riekkinen a

a Department of Neurology, University ofKuopio, PO Box 1627, FIN-70211 Kuopio, Finland b Baker Norton Pharmaceuticals, Inc., 50 Northwest 176 Street, Miami, FL 33169, USA

Accepted 28 November 1995

Abstract

The present study investigated the effects of repeated daily administration of a mixture containing free fatty acids (a-linolenic/lino- leic acid in a 1:4 ratio at 25 mg/kg i.p., Alzene ~) on medial septal (MS) lesion-induced impairment of water maze (WM) spatial reference and working memory or passive avoidance (PA) behavior in adult rats (250-275 g at the beginning of the study). Alzene treatment was started 7 days before initiation of behavioral testing (WM reference memory testing on treatment days: 7-9 and 28-30 + passive avoidance on treatment days 31-32; WM working memory testing on treatment days: 7-10 and 38-31 + passive avoidance testing on treatment days 32-33) and continued until the end of the study. Alzene improved WM reference memory (treatment days 7-9 and 28-30) and PA behavior (treatment days 31-32) as effectively as an anti-cholinesterase drug, tacrine 3 mg/kg (i.p.). However, in a separate group of MS-lesioned rats we observed that working memory (treatment days 7-10 and 28-31) was not improved by either Alzene or tacrine treatment. The present results suggest that Alzene treatment improves spatial reference memory and inhibitory avoidance in MS-lesioned rats.

Keywords: Alzene; Working and reference spatial memory; Passive avoidance; Medial septum; Essential fatty acid; Linolenic/linoleic acid

1. Introduct ion

Studies conducted using brain samples obtained from patients pathologically verified as having Alzheimer 's dis- ease (AD) have shown a consistent and marked loss of cholinergic cells in the nucleus basalis and medial septum [1,24]. The importance of this cholinergic cell loss for the development of dementia is supported by clinical [3,8,11] and experimental findings [2,23]. For example, treatment with tacrine, a cholinesterase inhibitor, to some extent alleviated clinical dementia of AD patients [3,8,11]. How- ever, the treatment response is modest at best and the severe dose-dependent side-effects limit the clinical use of tacrine [22]. Therefore, the development of novel, side-ef- fect free, drugs to treat the symptoms of AD would be a major advance. Furthermore, if the novel compounds have

* Corresponding author. Fax: (358) (71) 16-2048.

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01522-1

different mechanisms of action than tacrine, it is theoreti- cally possible that these drugs would be useful for add-on polytherapy with tacrine to increase the clinical efficacy. A combination of drugs may have synergistic effects in terms of efficacy and infra-additive effects on each other in terms of toxicity; i.e., an enhanced eff icacy/ toxici ty ratio.

One pharmacological model to study the efficacy of novel drugs to promote learning behavior is to investigate the water maze (WM) and passive avoidance (PA) perfor- mance modulating action of these compounds [6,7,10,14]. Several previous studies have found that cholinesterase inhibitors and muscarinic or nicotinic acetylcholine recep- tor agonists may improve performance of basal forebrain- lesioned rats in tests used to assess learning and memory [13,16]. For example, we observed that partial medial septal (MS)-lesions decreased hippocampal cholinergic ac- tivity and impaired WM and PA acquisition behavior [15-18]. Our recent studies have shown that cholinergic drugs may alleviate this MS lesion-induced behavioral defect [15,16]. First, tacrine had an inverted U-shaped dose

P. Riekkinen Jr. et al. / Brain Research 714 (1996) 118-124 119

response, at an optimal dose of 3 mg /kg it facilitated WM and PA behavior of MS-lesioned rats [15-18]. Second, nicotine, a nicotinic acetylcholine receptor agonist, at doses of 0.1 and 0.3 mg/kg stimulates WM and PA behavior of MS-lesioned rats [17].

In AD the levels of polyunsaturated fatty acids (PUFA) are reduced in frontal and hippocampal areas which may lead to a decrease in membrane fluidity and impair func- tion of membrane related ion channels, receptors and enzymes [19-21]. This impairment of membrane composi- tion in AD may be due to excess lipid peroxidant activity. Therefore, it is possible t]hat administration of essential fatty acids might restore membrane fluidity and improve neuronal transmission in AD brains [19].

Experimental studies have shown that manipulation of the type of fat in the diet can influence behavior. Initially, Lamptey et al. [12] reported an improvement of learning behavior in a simple Y-maze and higher levels of incorpo- ration of w3 fatty acids into brain glycerophosphatides on diet containing soybean oil as compared to a diet contain- ing safflower oil. Also soybean oil, in contrast to sun- flower oil, facilitated spati~d memory in Y-maze. Impor- tantly, soybean oil contains a considerably higher percent of a-linolenic acid than safflower or sunflower oil, which could lead to an increase in membrane fluidity [4,27]. These studies lead to the formulation of the hypothesis that the quantity of a-linolenic acid as well as the ratio of c~-linolenic acid to linoleic acid may be important for the central effects of diet fat manipulations [28,29]. This hy- pothesis was tested and supported by a study of Yehuda and Carasso [29,30] comparing a-linolenic and linoleic acid preparations with a ratio of fatty acids ranging from 1:3-1:6 on behavioral functions and brain plasma mem- brane lipids in young rats. That study found that fatty acid mixtures with a-linolenic and linoleic acid ratios between 1:3.5-1:5 significantly improved WM learning, increased pain threshold and protected from D-amphetamine treat- ment-induced hypothermia. Furthermore, the treatment de- creased cholesterol content and increased a-linolenic and linoleic acid content of brain plasma membranes. Yehuda and Carasso [29,30] proposed that the beneficial cognitive effects of free fatty acids result from modulation of the composition of membrane lipids as well as the membrane fluidity of brain neurons.

The aim of present experiment is to study if an a-lino- lenic acid and the linoleic acid preparation (Alzene ®) with an optimal ratio l:4 at a dose previously found most effective to facilitate spatial navigation could alleviate MS lesion-induced WM spatial reference/working memory and PA performance defect. Alzene was injected once every day for approximately 4 weeks and WM testing was conducted at two time points (beginning 7 or 28 days after the initiation of Alzene treatment) to investigate the time course of action of Alzene Furthermore, the effects of tacrine also were studied to estimate the magnitude of performance improvement induced by Alzene treatment.

2. Materials and methods

2.1. Animals

The rats (Han:Wistar; 250-275 g; total n = 72, 9 rats in every group) were housed three per cage in a controlled environment (20 _+ 2°C, humidity at 50-60%, light period 07.00-19.00 h). Food and water were available ad libitum. The body weight of rats was measured at the beginning and end of the experiment.

2.2. Brain lesioning

Brain lesioning was made on the same day as Alzene ® or Alzene vehicle treatments were initiated (see Fig. 1 for the treatment schedule and groups). Seven days later, behavioral testing was initiated. MS lesions were made as follows. The rats were deeply anesthetized with chloral hydrate (350 mg/kg, i.p.) and placed in a stereotaxic frame with the bregma and lambda in the horizontal plane. MS lesions (AP: 0.2 ram, ml: 0.0 rnm, d: - 7 . 0 mm, relative to the bregma) were performed with stainless steel electrodes (diameter 0.25 mm, 0.4 mm tip uninsulated) by passing anodal DC current (2 mA, 5 s) through the elec- trode. The sham lesioned groups were treated identically, but no current was applied.

W M R E F E R E N C E M E M O R Y + P A S S N E A V O I D A N C E

DRUG DAYS

ALZENE e 1 iJ 32

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Fig. 1. Alzene ® (25 mg/kg) /Alzene vehicle and tacrine (3 mg/kg)/ tacr ine vehicle treatment schedules are shown. The drugs were injected i.p. During behavioral training days, Alzene/Alzene vehicle and tacrine/tacrine vehicle were injected 3 h and 30 min before behavioral testing, respectively. Alzene/Alzene vehicle was injected also during other days as indicated in the figure. Tacrine/tacrine vehicle was not injected for the rats during passive avoidance testing day (i.e. day 32 or 33, see below). The following drug groups were used in the experiments testing the effects of study drug treatments on reference or working memory functioning and passive avoidance. Sham-lesion: Alzene vehicle + tacrine vehicle; control MS-lesioned: Alzene vehicle + tacrine vehicle; tacrine treated MS-lesioned: Alzene vehicle +tacrine 3 mg/kg; Alzene treated MS-lesioned: Alzene+tacrine vehicle. The number of rats in every group was 9. WM Reference memory + passive avoidance: WM behavioral testing was conducted in this experiment during two phases (days 7-9 and 28-30) and passive avoidance testing during days 31-32. WM Working memory + passive avoidance: WM behavioral testing was conducted in this experiment during two phases (days 7-10 and 28-31) and passive avoidance testing during days 32-33.

120 P. Riekkinen Jr. et al./Brain Research 714 (1996) 118-124

2.3. Drug treatment

Fig. 1 shows the drug treatment schedule. Alzene ® was composed of a-linolenic (0.92 g /ml) and

c~-linoleic (0.90 g /ml) free fatty acids in a ratio of 1:4 and was administered at a daily dose of 25 mg/kg (i.p.) of body weight. This mixture of free fatty acids, mineral oil and c~-tocopherol were mixed at a ratio of 0.25 m1:0.74 ml:0.01 ml (Ivax/Baker Norton Pharmaceuticals, Miami, FL, USA). Alzene vehicle was composed of mineral oil and ~-tocopherol at a ratio of 0.99 mi/0.01 ml (Ivax/Baker Norton Pharmaceuticals). During behavior training days Alzene and Alzene vehicle were injected 3 h before behavioral testing. Alzene and Alzene vehicle were injected at the same time of the day during those days, when behavior was not tested. These test substances were stored at + 4°C in the dark.

Tacrine (Sigma) was dissolved in 0.9% NaC1 and in- jected i.p. 30 min only before (2 ml/kg) WM training trials and the PA training trial. Tacrine was made fresh every testing day. During the behavioral training days, NaC1 0.9% was injected for those groups that did not receive tacrine.

2.4. Behavioral testing

Fig. 1 shows the behavioral testing schedule.

2.5. Water maze

A video camera was connected via an image analyzer to a computer system. This videotracking system monitored the swimming of the rats in the pool. The computer calculated the mean escape distance values. The group means of daily escape values were stored for statistical analysis of the effects of lesioning and drug treatment on water maze acquisition. The starting locations which were labelled north, south, east and west were located arbitrarily on the pool rim. The pool was divided into four quadrants of equal surface area. The platform was initially located in the south-east quadrant. Rats were placed in the water, with their nose pointing towards the wall, at one of the starting points in a random manner. If the rats did not find the hidden escape platform during the maximum time allowed, the tester placed them gently on the escape platform for 5 s.

Reference memory testing (Alzene/Alzene vehicle treatment day 7-9 and day 28-30). Testing consisted of 3 consecutive days (6 trials/day) of testing. Each rat was allowed 60 s to find the platform, with an interval of 30 s between the trials. On each trial, the rats were allowed to stay on the platform for 5 (s). The platform location was not changed during the training trials.

Working memory testing (Alzene / Alzene vehicle treat- ment day 7-10 and day 28-31). The testing of rats during the first two training days was identical to that described

above. However, on the third and fourth day of both of the testing periods, the platform location was altered. On the third day of testing the platform was moved to north-east quadrant. On the fourth day of testing the platform was once again located in the south-east quadrant.

2.6. Passive avoidance

A plexiglass PA box consisted of a bright and dark compartment. The compartments were divided by a guillo- tine door. The dark compartment had a metal grid floor. During the training trial (Alzene/Alzene vehicle treatment day 31 in the reference memory protocol; Alzene/Alzene vehicle treatment day 32 in the working memory protocol) the rats were placed into the bright compartment and 30 s later the guillotine door was opened. After the rats had entered the dark compartment, the guillotine door was closed and a shock was delivered (0.5 mA, 3 s). Next, the rats were moved back to their home cage. 24 h later the rats were again placed into the bright compartment. 30 s later, the guillotine door was opened and the latency to re-enter the dark compartment was measured (maximum duration 360 s). Short entry latencies indicated weak per- formance.

2.7. Biochemistry

The rats were decapitated rapidly 24 h after the last study drug injection. The hippocampi were bilaterally dis- sected on ice and stored at -74°C until assayed. The choline acetyltransferase (CHAT) assay [15] was used to analyze the degree of lesion-induced cholinergic defect in the hippocampus. The ChAT method has been described in detail previously. Four rats from every group were taken for biochemical analysis.

2.8. Statistics

The one-way ANOVA followed by Duncan's post-hoc multiple group comparison was used to analyze group differences. P < 0.05 was accepted as significant. A drug treatment-induced (tacrine or Alzene) decrease of escape distance values and increase of PA entry latencies of medial septal-lesioned rats was interpreted as an improve- ment of spatial navigation and avoidance performance, respectively.

3. Results

3.1. Reference memory testing in water maze

3.1.1. Treatment day 7 -9 Fig. 2 shows the escape distance values measured dur-

ing treatment day 7-9. On Day 7 all the MS-lesioned groups were impaired compared with the sham-lesioned

P. Riekkinen Jr. et al. /Brain Research 714 (1996) 118-124 121

(overall effect: F = 18.09, P < 0.0001; P < 0.05 for all comparisons against the controls). However, the control MS-lesion group was more impaired than Alzene ®- or tacrine 3 mg/kg- t rea ted rats ( P < 0.05, in both compar- isons).

On Day 8, significant differences were found between the groups (overall effect: F = 12.12, P < 0 . 0 0 0 1 ) : tacrine-treated and control MS-lesion rats were impaired compared with the sham-lesioned ( P < 0.05, for both com- parisons). However, control MS-lesion rats were still more impaired than the tacrine-treated group ( P < 0.05).

On Day 9 (overall effect: F = 8.54, P = 0.0003), all the MS-lesioned groups were impaired compared with the sham-lesioned ( P < 0.05, for all comparisons). However, control MS-lesion rats were more impaired than Alzene- or tacrine-treated rats ( P < 0.05, for both comparisons).

3.1.2. Treatment day 28-:~0 Fig. 3 shows the data measured during Day 28-30. On

Day 28 marked group differences were found in the analy- sis of WM data measured (overall effect: F = 17.26, P < 0.0001): all the MS-lesioned rats were impaired ( P < 0.05, for all comparisons). However, tacrine-treated rats and Alzene-treated rats were less impaired than the control MS-lesion group ( P < 0.05, for both comparisons).

On Day 29, all MS-lesioned groups were significantly impaired (overall effect: F --- 9.73, P = 0.0001; P < 0.05 for all comparisons). However, the control MS-lesion rats

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Fig. 2. The effect of tacrine and Alzene on water maze reference memory functioning of medial septal-lesioned rats during Days 7-9. Alzene treatment was started on Day 1. Control = sham-lesioned; Vehicle = vehicle-treated lesioned rats; Alzene=Alzene-treated lesioned rats; Tacrine = tacrine-treated lesioned rats.

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were the most impaired group as the escape distance values of that group were longer than those of the tacrine- or Alzene-treated groups ( P < 0.05).

On Day 30, significant overall group effect was found on the escape distance values ( F = 9.17, P = 0 . 0 0 0 2 ) . Tacrine-treated rats and control MS-lesion rats were im- paired ( P < 0.05, for both comparisons). However, the tacrine-treated group was significantly better than control MS-lesion rats on the last day of WM testing ( P < 0.05).

3.1.3. Passive avoidance A marked overall effect was found in the analysis of

passive avoidance entry latencies during the testing trial ( F = 21.24, P < 0.0001)(Table 1, Part A). All of the MS-lesioned groups were impaired ( P < 0.05, for all com- parisons), but control MS-lesion rats were more impaired than the other two groups of MS-lesioned rats ( P < 0.05, for both comparisons).

3.2. Working memory testing in water maze

3.2.1. Treatment day 7-10 Fig. 4 shows the escape distance values measured dur-

ing Day 7-10. As was observed in the study investigating the effects of Alzene and tacrine on reference memory functioning, MS-lesioning disrupted WM behavior. On Day 7 and 8 all the MS-lesioned groups were impaired compared with the sham-lesioned (Day 7 overall effect: F = 23.44, P < 0.0001 and Day 8 overall effect: F =

122 P. Riekkinen Jr. et al. / Brain Research 714 (1996) 118-124

Table 1 Effects of daily treatment with a-linolenic acid and linoleic acid prepara- tion at a ratio 1:4 (Alzene ®) at 25 mg/kg i.p. and single pretraining treatment with tacrine 3 mg/kg on medial septal lesion-induced passive avoidance failure

Group Latency

PART A Control 350 + 14 Vehicle 186 + 54 * Alzene 299 + 41 * + Tacrine 286 + 57 * ÷ PART B Control 333 + 23 Vehicle 167 + 36 * Alzene 254 + 42 * ÷ Tacrine 271 +51 * ÷

Parts A and B show the results of the study conducted using the rats involved in reference memory and working memory WM testing, respec- tively. The groups in Part A received Alzene or Alzene vehicle for 32 days. Passive avoidance training and testing trials were conducted on the 31st and 32nd Alzene/Alzene vehicle treatment days. The groups in Part B received Alzene or Alzene vehicle for 33 days. Passive avoidance training and testing trials were conducted on the 32nd and 33rd Alzene/Alzene vehicle treatment days. Passive avoidance testing trial entry latencies (s). The values are group mean + S.D. * P <0.05 vs. controls, + P <0.05 vs. controls/vehicle. One-way ANOVA, Duncan's post-hoe multiple group comparison.

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Fig. 5. The effect of tacrine and Alzene on water maze reference memory functioning of medial septal-lesioned rats during Days 28-31. Alzene treatment was started on Day 1. For definition of Groups, see legend to Fig. 2.

47.95, P < 0.0001; P < 0.05 for all comparisons against the controls). However, the control MS-lesion group was more impaired than Alzene- or tacrine 3 mg/kg-treated rats (P < 0.05, in both comparisons). The tacrine-treated

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D A Y Fig. 4. The effect of tacrine and Alzene on water maze working memory functioning of medial septal-lesioned rats during Days 7-10. Alzene treatment was started on Day 1. For definition of Groups, see legend to Fig. 2.

MS-lesioned group was slightly and non-significantly bet- ter on Day 7 and 8 than the Alzene-treated lesioned rats (P > 0.05).

On Day 9 (overall effect: F = 36.10, P < 0.0001) all the MS-lesioned groups were impaired compared with the sham-lesioned ( P < 0 . 0 5 , for all comparisons). Both Alzene and tacrine treatments failed to significantly facili- tate spatial navigation (P > 0.05, for both comparisons).

On Day 10 (overall effect: F = 27.29, P < 0.0001) all the MS-lesioned groups were impaired and the Alzene- and tacrine-treated rats were only non-significantly better than the control MS-lesion rats (P > 0.05).

3.2.2. Treatment day 28-31 Fig. 5 shows the data measured during Day 28-31.

Again, marked overall group differences were found in the analysis of WM data measured during Day 28 (overall effect: F = 67.20, P < 0.0001) and Day 29 (overall effect: F = 37.17, P < 0.0001): all the MS-lesioned rats were impaired (P < 0.05, for all comparisons). However, tacrine- and Alzene-treated rats were less impaired than the control MS-lesion group (P < 0.05, for all compar- isons).

On Day 30 (F = 27.03, P < 0.0001) and 31 (F = 37.52, P < 0.0001), significant overall group effects were found on the escape distance values. Alzene- and tacrine-treated rats did not perform significantly better than the control MS-lesion rats (P > 0.05).

P. Riekkinen Jr. et al. / Brain Research 714 (1996) 118-124 123

3.2.3. Pass ive avoidance

Again a marked overall effect was found in the analysis of passive avoidance entry latencies ( F = 27.35, P < 0.0001)(Table 1, Part B). All of the MS-lesioned groups were impaired (P < 0.05, for all comparisons), but control MS-lesion rats were more impaired than the other two groups of MS-lesioned rats (P < 0.05, for both compar- isons).

3.3. Biochemis try

MS lesions decreased hippocampal ChAT activity ( F = 37.22, P < 0.001). All of the three different treatment groups (Alzene vehicle + tacrine vehicle; Alzene + tacrine vehicle; tacrine + Alzene vehicle) of the MS-lesioned rats had equally low ChAT values (P > 0.05) (mean ___ S.D., nmol /mg protein/min: controls: 1.18 + 0.08; control MS-lesion: 0.45 ___ 0.11; MS-lesion + tacrine: 0.50 __+ 0.12; MS-lesion + Alzene: 0.42 ___ 0.13).

4. Discussion

The present results confirmed earlier findings showing that daily pretraining treatment with an optimal dose of tacrine (3 mg/kg) improved reference memory and PA behavior of MS-lesioned rats, but had no effect on working memory performance [15]. More interesting and novel was the finding that repeated &fily administration of the a-lino- lenic and linoleic acid preparation at a ratio 1:4 at 25 mg/kg i.p. improved WM reference memory and PA behavior of MS-lesioned rats as effectively as tacrine. Furthermore, tolerance did not develop to therapeutic ef- fect of the experimental fatty acid mixture during a 4-week repeated daily treatment.

The present study was; not designed to examine the mechanism of action of Alzene ®, but to investigate if the drug has any effect on spatial navigation and PA behavior of MS-lesioned rats, and to compare the magnitude of the treatment response with an optimal dose of the anti- cholinesterase, tacrine. Nevertheless, some important re- suits of the present study merit discussion.

The present results show that Alzene treatment can improve reference memor.¢ and PA behavior in rats sub- jected to partial MS lesions. This may be explained by different pharmacological and neurobiological theories. First, it could be proposed that Alzene stimulates the activity of the partially damaged cholinergic projection from the septal area to the hippocampus. This hypothesis is not, however, supported by the present results as we found that the degree of ChAT depletion in the hippocampus of MS-lesioned rats treated with Alzene did not differ from control or tacrine-treated lesioned rats. The lack of an effect on ChAT activity suggests that Alzene treatment has no major effect on tonic activity of acetylcholine fibers. However, it is still possible that Alzene treatment may

have modulated presynaptic cholinergic activity, but ChAT enzyme activity may not be a sensitive indicator of such an effect. Interestingly, a previous study of Yamamoto et al. [25,26] described an altemative possibility for the cogni- tive enhancing effect of fatty acids. They [25] found that dietary supplementation with a mixture of a-linolenic acid/a-linoleic acid at a ratio of 1:5 modulated neuronal membrane lipid composition, improved learning and en- hanced survival time in aged rats. Indeed, Yehuda and Carasso [29] showed that Alzene treatment decreases cholesterol content and increases a-linolenic acid and linoleic acid content in the hippocampal plasma mem- brane. Interestingly, an in vitro increase of membrane cholesterol content decreased the effect of cholinergic receptor stimulation on functioning of hippocampal pyra- midal neurons [5]. Thus, Alzene treatment can alter neu- ronal plasma membrane composition of cholesterol and fatty acids which may facilitate the functioning of mem- brane spanning receptors [9,25], such as cholinergic recep- tors, and enhance the post-synaptic response of acetyl- choline in hippocampus resulting in improved WM learn- ing and PA behavior. Second, the electrolytic lesioning method used in the present study have also destroyed fibers of passage and non-cholinergic cells in the lesioned area. Therefore, Alzene may have alleviated that compo- nent of memory failure caused by lesion of these non- cholinergic fibers and neurons [9], since the neuronal membrane function modification is likely to affect a vari- ety of receptor systems [20]. This issue could be investi- gated by studying the effects of treatment with Alzene on WM performance failure induced by cholinergic antago- nist, such as scopolamine.

The lack of an effect of tacrine treatment on WM working memory failure in MS-lesioned rats may not be explained simply in terms of an inadequate dose. In our earlier studies we have found that smaller 1 mg /kg dose of tacrine was ineffective, and at higher 5 mg/kg dose of tacrine rats could not swim in the pool [15-18]. Therefore, it is possible that tacrine may not fully restore cholinergic transmission in the hippocampus and/or that tacrine alle- viates only that part of the lesion-induced defect resulting from cholinergic cell loss. Similarly, the ineffectiveness of Alzene to significantly improve behavior during working memory testing in WM may be caused by the selection of a suboptimal dose as only one dose was examined in this model. However, an earlier dose response study described that 25 mg/kg was the best dose of Alzene to facilitate WM learning in rats [29]. Therefore, we propose that Alzene treatment does not seem to facilitate the function- ing of these brain areas important for working memory performance in MS-lesioned rats.

In conclusion, the present study supports previous evi- dence indicating that modulation of dietary fatty acids may affect cognitive functioning [12,25-30]. Therefore, treat- ment with an optimal dietary supplement composed of a mixture of a-linolenic and a-linoleic acid may be an

124 P. Riekkinen Jr. et al. / Brain Research 714 (1996) 118-124

effective therapeutic approach to alleviate age- and AD-re- lated neuronal membrane defects and cognitive failure. Furthermore, the potential of combined tacrine and Alzene treatments to facilitate cognitive functioning in animal cognition models should be investigated further.

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